The invention relates to methods to synthesize polyketides which are novel using modified modular polyketides synthases (PKS) which cannot utilize a natural first module starter unit.
Modular polyketide syntheses are typified by the organization of deoxyerythronolide B synthase (DEBS) which produces xcex2-deoxyerythronolide B (6-dEB) the parent macrolactone of the broad spectrum antibiotic erythromycin. DEBS consists of three large polypeptides each containing about 10 distinctive active sites. FIG. 1 shows, diagrammatically, the nature of the three DEBS modules encoded by the three genes eryAI, eryAII and eryAIII.
Various strategies have been suggested for genetic manipulation of PKS to produce novel polyketides. New polyketides have been generated through module deletion (Kao, C. M. et al., J. Am. Chem. Soc. (1995) 117:9105-9106; Kao, C. M. et al, J. Am. Chem. Soc. (1996) 118:9184-9185). Also reported to provide novel polyketides are loss of function mutagenesis within reductive domains (Donadio, S. et al., Science (1991) 252:675-679; Donadio, S. et al, Proc. Natl. Acad Sci. USA (1993) 90:7119-7123; Bedford, D. et al., Chem. Biol. (1996) 3:827-831) and replacement of acyl transferase domains to alter starter or extender unit specificity (Oliynyk, M et al., Chem. Biol. (1996) 3:833-839; Kuhstoss, S. et al., Gene (1996) 183:231-236), as well as gain of function mutagenesis to introduce new catalytic activities within existing modules (McDaniel, R. et al., J. Am. Chem. Soc. (1997) in press). In some of these reports, downstream enzymes in the polyketide pathway have been shown to process non-natural intermediates. However, these methods for providing novel polyketides suffer from the disadvantages of requiring investment in cloning and DNA sequencing, the systems used being limited to producer organisms for which gene replacement techniques have been developed, primer and extender units that can only be derived from metabolically accessible CoA thioesters, and the fact that only limited auxiliary catalytic functions can be employed.
The DEBS system in particular has been shown to accept non-natural primer units such as acetyl and butyryl-CoA (Wiesmann, KEH et al, Chem. Biol. (1995) 2:583-589; Pieper, R. et al, J. Am. Chem. Soc. (1995) 117:11373-11374) as well as N-acetylcysteamine (NAC) thioesters of their corresponding diketides (Pieper, R. et al., Nature (1995) 378:263-266). However, it has become clear that even though such unnatural substrates can be utilized, competition from the natural starter unit has drastically lowered yield. Even if starter units are not supplied artificially, they can be inherently generated from decarboxylation of the methylmalonyl extender units employed by the DEBS system (Pieper, R. et al., Biochemistry (1996) 35:2054-2060; Pieper, R. et al., Biochemistry (1997) 36:1846-1851).
Accordingly, it would be advantageous to provide a mutant form of the modular polyketide synthesis system which cannot employ the natural starter unit. Such systems can be induced to make novel polyketides by supplying, instead, a suitable diketide as an NAC thioester or other suitable thioester. Mutations have been made in the past to eliminate the competition from natural materials (Daum, S. J. et al., Ann. Rev. Microbiol. (1979) 33:241-265). Novel avermectin derivatives have been synthesized using a randomly generated mutant strain of the avermectin producing organism (Dutton, C. J. et al., Tetrahedron Letters (1994) 35:327-330; Dutton, C. J. et al., J. Antibiot. (1991) 44:357-365). This strategy is, however, not generally applicable due to inefficiencies in both mutagenesis and incorporation of the substrates.
Thus, there is a need for a more efficient system to prepare novel polyketides by inhibiting competitive production of the natural product.
The invention is directed to methods to prepare novel polyketides using modified modular polyketide synthase systems wherein directed modification incapacitates the system from using its natural starting material. Novel polyketides can then be synthesized by overriding the starter module and supplying a variety of suitable diketide substrates.
Thus, in one aspect, the invention is directed to a method to prepare a novel polyketide which method comprises providing a thioester diketide substrate to a modular PKS comprising at least two modules under conditions wherein said substrate is converted by said modular PKS to a product polyketide, wherein said PKS has been modified to prevent its utilization of the native starter unit. In other aspects, the invention is directed to the modified modular PKS which is disarmed with respect to utilization of the native starter substrate supplying the initial two carbon unit, and to suitable cells modified to contain this disarmed PKS. The invention is further directed to recombinant materials for production of the modified PKS and to the novel polyketides produced by this system.